Recent evidence suggests that there is a link between enhanced expression of uncoupling protein 2 (UCP2) and development of type II diabetes. A major focus of this proposal is to investigate UCP2 function and pancreatic Beta-cell bioenergetics in an effort to clarify this connection. We will employ our yeast expression system for UCP2 to perform the reference experiments, which are required to confirm that a particular property is inherent to UCP2. Thus, experiments will be performed in vitro with recombinant yeast-expressed UCP2 and also with mitochondria isolated from pancreatic 13-cells. The Specific Aims of the proposal are: To study substrate requirements of recombinant yeast-expressed UCP2 to determine whether UCP2-mediated proton transport has a particular requirement for fatty acid chain and saturation. To study nucleotide binding and inhibition of UCP2 and to identify other natural and artificial inhibitors of UCP2. To study the bioenergetics of mitochondria isolated from pancreatic beta-cells that have been treated or untreated with PPAR agonists. Aims 1 and 2 will employ reconstitutions and measurements of proton flux. They will also include a test of the hypothesis that fatty acid-induced proton transport by UCP2 exhibits an absolute requirement for coenzyme Q10. Aim 3 will utilize sensitive and precise measurements of oxygen consumption to detect UCP2 activity in Beta-cell mitochondria. We will attempt to distinguish between the contributions of increased UCP2 expression and altered biochemical regulation of UCP2 in mitochondria from treated and untreated cells.